In the last decades considerable attention has been directed by the scientific community to the derivation of cells, in particular stem cells (SCs) maintaining the ability to differentiate into specific tissue cells. This interest has been fueled by a strong need and desire to develop novel tissue and cell replacement approaches to heal the ailing body and reestablish cells and functions of afflicted and damaged tissues and parts of the body.
SCs are found in all multicellular organisms. They have the capacity to self-renew and to develop into different specialized cells. Mammalian stem cells are categorized into two general types, embryonic stem cells (ES cells) found during early embryonic development and adult stem cells (ASCs) found in tissues of the organism at later stages and throughout life.
ASCs, also called somatic stem cells; possess the same basic characteristics of all SCs. Namely they are capable of indefinite self-renewal, sometimes during an animal's entire life, and differentiation into specialized cell types. In mammals, the key tasks of ASCs are to sustain and heal the tissues in which where they are located. They remain as undifferentiated cells that may be activated upon tissue damage or other conditions to form multiple cell types and repair tissue damage (Friedenstein A et al. (1974). Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 2:83-92).
Additionally, ASCs secrete a large number of bioactive molecules within the tissue which act by modulating the inflammatory response of cells microenvironment, controlling angiogenesis and proliferation of cells involved in tissue repair process, and are therefore also known as pericytes (Caplan A I (2009). Why are MSCs therapeutic? New data: new insight. J Pathol. 217:318-324).
However ASCs have limited capacity for differentiation in comparison with pluripotent embryonic stem cells, and can usually differentiate to form only specific cell types of their tissue of origin. In contrast to embryonic stem cells, adult stem cells are not able to build a whole organism.
It is ES cells, existing during early stages of embryonic development (e.g. blastocyst stage), which possess truly high plasticity and pluripotency enabling them to differentiate to cells derived from all germ layers (Evans, M., and Kaufman, M. (1981) Establishment in culture of pluripotent cells from mouse embryos. Nature 292, 154-6). Upon reintroduction into a blastocyst, ES cells may recommence their regular development and may colonize the germinative tissues and additional embryo tissues.
Cultured ES cells may conserve an undifferentiated state over a long period and numerous cell divisions. On the other hand cultured ES cells can also be induced to differentiate. For instance, when cultivated in suspension, ES cells cultured in suspension form three-dimensional aggregates of pluripotent ES cells known as “embryoid bodies” (Ebs), which undergo cell specification to cells of all three germ lines (Ling, V., and Neben, S. (1997) In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies. J. Cell Physiol. 171, 104-5).
In view of their high pluripotency embryonic stem cells would theoretically be ideal cells to use in a large number of novel medical applications. However, the use of ES cells has generated a lot of controversy and significant ethical and legal concerns hinder their use in stem cell therapy. Treatment based on ASCs is much less controversial as it does not necessitate destruction of human embryos. Additionally ASCs do not produce teratomas and are of low immunogenicity.
For these reasons non-embryonic ASCs have become the focus of much research and work. After isolation and in vitro expansion, ASCs are known as mesenchymal stem cells (MSCs). MSCs are multipotent and preserve their in vivo ability to differentiate in vitro into several cell types such as bone, cartilage and muscle.
Regrettably, the challenges for practical use of adult stem cells in therapy are numerous. They are found only in infinitesimal numbers and it is extremely difficult to isolate them in useful amounts. Moreover their propagative capacity is relatively low and they may contain various DNA aberrations.
Another inherent difficulty and challenge for using MSCs in therapy is that their differentiation capacity is greatly dependent on suitable isolation and culture conditions. It is thus clear that in order for MSCs to fulfill their potential to treat a large number of devastating diseases it is necessary to achieve control over large scale production settings.
There are different methods of scaling up, but as a general rule, because of stem cells' inherent sensitivity to their environment, large-scale processes need to remain substantially similar to their original small-scale process for maintaining critical quality attributes. Davie, N. L. et al., (2012) Streamlining Cell Therapy Manufacture from Clinical to Commercial Scale. BioProcess International 10(3), 24-29. Thus, if cells grown for early clinical studies are cultured on planar surfaces (such as a T-flasks or trays), then on reaching larger-scale operations, those cells will need to be cultured similarly. But that may be difficult to implement and limit production capacity later on. Likewise, if a desired end point of a scaled-up process is to culture cells on microcarriers, then the product used in early studies ideally should have also been produced using suspension culture. If not, final product cells will probably not to be comparable to those later used in the clinic.
Even historically predictable scale-up strategies (e.g., from small to larger Nunc Cell Factory) can impart some unwanted cellular changes, such as the loss or gain of a certain marker or the halted or activated secretion of a particular cytokine. However, it is sometimes the case that such changes resulting from scaling methodologies are irrelevant to a product's clinical function. If it can be proven that the given changes do not affect the proposed mechanism of action or cause adverse side effects, then the new process is likely to be accepted by regulators. Agencies recognize this, which is why products need to be demonstrated as “comparable” and not “identical.”
Indeed, improved methods for isolation and propagation of undifferentiated MSCs are considered vital for the development of novel adult stem cell therapy and regenerative medicine.
A number of processes are previously known for obtaining large numbers of MSCs from different tissues while preserving their capability for differentiation. (Kuehle, I., and Goodell, M. A. (2002). The therapeutic potential of stem cells from adults, B.M.J. 325, 372-376; Pittenger, M. F., and Martin, B J. (2004) and mesenchymal stem cells and their potential as cardiac therapeutics. Circ. Res. 95, 9-20).
Nevertheless, these methods result in isolation of MSCs having restricted differentiation ability, giving rise to only a limited variety of cell types. Moreover since the number of ASCs in tissues is very low these methods result in non-homogeneous cultures, containing large numbers of cells, which are not stem cells.
The main sources of MSCs currently used in medicine are the bone marrow and the umbilical cord. However, the isolation of pure populations of pluripotent SCs and in large amounts from these tissues is difficult. Alternative sources for the isolation of highly pluripotent and homogeneous stem cells are required, which will significantly increase the treatment efficacy of several diseases.
One such likely source includes MSCs derived from teeth and dental tissues. Teeth present a readily accessible source for obtaining MSCs useful for tissue regeneration and repair. Similar to other organs in the human body, the teeth and their surrounding tissues are composed by mixed populations of cells, which include multipotent MSCs/pericytes, progenitor and differentiated cells (Arthur et al. (2008). Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26: 1787-1795; Giordano G et al. (2011) Stem cells from oral niches: a review. Ann Stomatol (Roma) 2:3-8).
To conduct stem cell therapies significant in vitro expansion of stem cells is necessary in order to generate sufficient quantities of these cells to treat human disease. At the present time, there is no known reliable way to efficiently generate large numbers of relatively pure dental pulp stem cells populations in culture.
One of the reasons for this is that the regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation (Wagner W et al. (2009). Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS One 4:e5846).
Another important reason for the difficulty in large-scale production of dental pulp stem cells is that the expansion process itself induces senescence of stem cells and the loss of their stemness as shown by a decline in proliferative and differentiation capacity. Baxter et al. demonstrated the effect of in vitro expansion on the replicative capacity of MSCs by correlating their rate of telomere loss during in vitro expansion with their behavior in vivo. They showed that even protocols that involve minimal expansion induce a rapid aging of MSCs, with losses equivalent to about half their total replicative lifespan. (Baxter M A et al. (2004) Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22, 675-682).
Moreover, prolonged culture of stem cells correlates with increased probability of genetic changes, which is detrimental to their safe use in clinical trials and in future therapies. For instance Wang et al. suggest that animals and cell culture differ in their ability to carry out genomic rearrangements as a means of maintaining telomere integrity when telomeres become critically shortened. (Wang Yet al. (2005). An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres. Proc Natl Acad Sci USA. July 19; 102(29):10256-60).
It is evident that the key to successful stem cell therapy is related to the process of harvesting of the cells and their expansion. This process must ideally allow for the production of very large amounts of a patient's own stem cells while at the same time maintaining their maximal capacity for differentiation. In this way patients who cryopreserve stem cells from their deciduous teeth as children or provide stem cells from their wisdom teeth or molars at later stages of life could benefit from the full therapeutic capacity of these precious cells.
It is very important to develop protocols for DSCs that will reduce the risk of tumorgenicity due to genetic abnormalities acquired during multiple passaging ASCs. It was shown in several preclinical studies that the success and efficiency of stem cells transplantation depends on the stem cells type, its proliferative and migration capacity and the site of injection.
Therefore, methods for the unlimited and consistent large scale production of highly potent ASCs of excellent quality and reduced risk of tumorgenicity at early passages derived from the same donor are desirable for the development of adult stem cell therapy.